Prokaryotic diacylglycerol kinase (DAGK) is an integral membrane protein (IMP) that functions as a 40 kDa homotrimer with 9 transmembrane helices. DAGK plays an essential role in the physiology of many bacteria and is a potential target for antimicrobial agents. While it is a structurally unique kinase, DAGK may represent an entire family of uncharacterized membrane proteins and is also an ideal system for mechanistic studies of membrane-integral biocatalysis. When correctly folded, DAGK is generally very stable. However, numerous mutant forms of this protein exhibit a tendency to misfold into kinetically-trapped conformations. DAGK is therefore an excellent system in which to examine the biophysical properties underlying membrane protein folding and misfolding, a matter of pressing medical relevance. The specific aims of the upcoming phase of this project are: Aim 1: Conduct solution NMR-based structural studies. (A) Enhance the resolution of our nearly completed NMR-based 3-D structure of DAGK by collecting additional data restraints. (B) Use NMR methods to map the nucleotide and diacylglycerol substrate binding sites. (C) Determine whether DAGK undergoes conformational changes on formation of binary and/or ternary complexes with substrates. This will include determination of the structure of DAGK's ternary complex with diacylglycerol and a non- hydrolyzable ATP analog. Aim 2: Conduct studies of folding and misfolding. (A) Follow up on key recent observations regarding DAGK folding and misfolding under conditions of in vitro denaturant-to-model membrane folding assays. (B) Identity factors that lead to DAGK misfolding after the point in the folding pathway at which membrane integration has been attained. Aim 3: Conduct mechanistic studies to determine the specific roles played by DAGK's active site residues in substrate binding and phosphoryl transfer. Of particular interest is to correlate the mechanistic insight from this aim with structural information from Aim 1 to determine how DAGK selectively binds its lipid substrates, diacylglycerol and phosphatidic acid, without competitive inhibition by other membrane lipids. [unreadable] [unreadable] [unreadable]